Biomedical subjects

Axel Seltsam

Publications and source records attributed to Axel Seltsam.

13 recordsLinked to original sources

PubMed2006

Class-, gene-, and group-specific HLA silencing by lentiviral shRNA delivery.

HLA incompatibility is the most relevant immunologic barrier to cell-based therapies. Improvement of histocompatibility is essential to achieving better survival of allogeneic cells in the foreign organism. RNA interference technology can be used to selectively and stably reduce cellular HLA class I expression. In the present study, we designed small interfering RNA (siRNA) molecules that target either beta2-microglobulin (beta2m) or HLA-A heavy chain transcripts and identified sensitive sites on the target RNAs using an in vitro transcription/translation (IVTT) system. Transfection of siRNA into B-lymphocyte cell lines (B-LCLs) resulted in specific reduction of HLA class I or HLA-A antigen expression by 79% at the mRNA and protein levels. An allele-specific HLA silencing rate of 65% was achieved in a B-LCL heterozygous for HLA-A*24,*68 allospecificities using HLA-A*68-specific siRNA. Lentiviral delivery of short hairpin RNA into HeLa and B-LCL cells resulted in selective and permanent silencing of HLA class I or HLA-A by up to 90% even under inflammatory conditions. In cytotoxicity and proliferation assays, it was demonstrated that HLA class I knockdown was effective in preventing antibody-mediated cell lysis and CD8+ T cell response, while the residual HLA expression in HLA-silenced cells was protective against NK-cell-mediated lysis. The present data strongly suggest that silencing of HLA expression in a class-, gene-, and group-specific manner is an effective approach that may provide a new basis for developing new immunotherapies in the field of regenerative medicine.

B-Lymphocytes↗

PubMed2006

A weak blood group A phenotype caused by a translation-initiator mutation in the ABO gene.

BACKGROUND: Weak blood group A and B phenotypes are correlated with ABO glycosyltransferases exhibiting single-amino-acid changes and/or C-terminal modifications. STUDY DESIGN AND METHODS: A healthy donor diagnosed as having weak A antigen expression and his two children were subjected to extensive ABO typing. HeLa cells were used to transfect ABO expression plasmids. RESULTS: The donor's red blood cells were type A(weak)B and his serum sample contained weakly reactive anti-A(1) antibodies. A single T>C transition identified at the +2 position of the start codon of an ABO*A101 allele predicted the disruption of this methionine codon. In the transfection studies, a significant reduction of A activity was observed on HeLa cells transfected with a plasmid containing the variant ABO*A allele. Coexpression of the respective antithetical ABO*B101 wild-type construct further reduced cell surface A antigen expression. Similar expression results were obtained with ABO constructs in which the Met(1) start codon and five alternative start sites at codons 20, 26, 43, 53, and 69 had successively been interrupted. CONCLUSION: The donor's weak blood group A phenotype most likely resulted from expression of an N-truncated A transferase triggered by alternative translation start sites in the transmembrane domain or stem region.

ABO Blood-Group System↗

PubMed2005

Nondeletional ABO*O alleles frequently cause blood donor typing problems.

BACKGROUND: Difficulties in the demonstration of expected isoagglutinins is a common problem in ABO reverse typing. Some nondeletional ABO*O alleles have been shown to encode for the expression of minimal amounts of A antigen, resulting in very weak anti-A activity in some cases. It is unknown whether minor problems with ABO reverse typing are related to specific ABO*O alleles. STUDY DESIGN AND METHODS: Among 2196 blood group O red cell (RBC) donations, the ABO alleles of those donations in which the isoagglutinins were incorrectly identified were analyzed with an autoanalyzer. The presence of nondeletional ABO alleles was determined by sequence-specific priming and sequencing. RESULTS: Fifty (2.3%) of the group O RBC donations tested had to be typed manually because of isoagglutinin detection problems in automated typing: reduced anti-A activity was observed in 45 cases, reduced anti-B activity in 4 cases, and variably reduced isoagglutinin activity in 1 case. The nondeletional ABO*O alleles ABO*O03 and ABO*Aw08 were implicated in 38 of these 50 cases (1.7% of all blood group O donors). The remaining samples, including those with reduced anti-B activities, were homozygous for deletional ABO*O alleles. CONCLUSION: Nondeletional ABO*O alleles are the most frequent cause of isoagglutinin detection problems in blood group O donors.

ABO Blood-Group System↗

PubMed2005

Missense mutations outside the catalytic domain of the ABO glycosyltransferase can cause weak blood group A and B phenotypes.

BACKGROUND: Only little is known about the impact of amino acid substitutions outside an enzyme's active site on A and B transferase activity. STUDY DESIGN AND METHODS: A panel of blood group A- and B-specific plasmids containing the six known missense mutations of the coding sequence upstream of exon 6 of the ABO gene were constructed. HeLa cells were used to transfect ABO expression plasmids. RESULTS: Expression of ABO variants containing single or multiple missense mutations in HeLa cells resulted in a significant decrease in the percentage of antigen-expressing cells (up to 29%) and in mean fluorescence intensity (MFI; up to 50%) compared to transfection with ABO*A101 or ABO*B101. Coexpression of the respective antithetical wild-type construct (ABO*A101 and ABO*B101, respectively) further reduced cell surface expression of variant ABO constructs in regard to the percentage of expressing cells (up to 53% decrease) and MFI (up to 59% decrease). CONCLUSION: Weak A and B subgroups can arise from transferases with amino acid changes in the N-terminal domain, particularly in AB phenotypes, where normal A1 or B1 glycosyltransferases compete for the same substrates.

ABO Blood-Group System↗

PubMed2005

Nondeletional ABO*O alleles express weak blood group A phenotypes.

BACKGROUND: Owing to a single-base deletion, the vast majority of ABO*O alleles encode for a truncated and catalytically inactive ABO glycosyltransferase, leading to the generation of a premature stop codon. Less frequent nondeletional ABO*O alleles such as ABO*O03, in contrast, have nonsynonymous mutations that may abolish the protein's enzyme activity by altering its sugar-binding site. STUDY DESIGN AND METHODS: Extensive ABO phenotyping and genotyping were performed in healthy blood group O donors with weak anti-A isoagglutinins and their relatives as well as in blood group O donors selected for the presence of ABO*O03. HeLa cells were used to transfect ABO expression plasmids. RESULTS: Donors or relatives carrying ABO*O03 and/or its rare variant ABO*Aw08 in homozygous (n = 2) or heterozygous (n = 14) form showed weak A antigen expression detectable only by adsorption-elution (n = 15) or by monoclonal anti-A typing (n = 1). The serum samples of most donors (n = 13) contained weak anti-A; in the remaining donors, anti-A isoagglutinin reactivity was in the normal range. In the transfection studies, weak A antigen expression on HeLa cells transfected with plasmids containing ABO*O03 or ABO*Aw08 expression constructs was detectable only by adsorption-elution. CONCLUSION: The data provide evidence that nondeletional ABO*O03-like alleles produce detectable amounts of A antigens.

ABO Blood-Group System↗

PubMed2005

ABO glycosyltransferases as potential source of minor histocompatibility antigens in allogeneic peripheral blood progenitor cell transplantation.

BACKGROUND: Most studies indicate that the incidence of graft-versus-host disease (GVHD) is not increased in ABO-mismatched allogeneic peripheral blood progenitor cell transplantation. These studies exclusively looked at ABO phenotypes without considering the fact that different genotypes hide behind identical phenotypes that encode for different sets of glycosyltransferases, thus providing a source for minor histocompatibility antigens (mHags). STUDY DESIGN AND METHODS: Therefore, whether peptides derived from ABO glycosyltransferases are capable of stimulating peptide-specific T cells was investigated. T-cell responses were identified by measuring intracellular interleukin-2 expression. RESULTS: Individuals with ABO genotypes encoding glycosyltransferases lacking the peptide sequences used for stimulation showed T-cell responses, whereas those expressing glycosyltransferases containing the respective peptide sequences proved to be tolerant, indicating that ABO peptides are allogeneic and may act as mHags. Interestingly, even ABO*O individuals were tolerant to O glycosyltransferase-derived peptides, which strongly suggests that truncated O transferases are expressed. CONCLUSION: Considering allelic ABO sequences, at least 15 percent of all phenotypically ABO-matched transplant pairs can be expected to have genotype constellations relevant to GVHD. Therefore, the genotype behind the ABO blood group phenotype should be considered to answer the question of whether ABO mismatch is a risk factor of GVHD.

ABO Blood-Group System↗

PubMed2005

Down-regulation of the IGF-2/H19 locus during normal and malignant hematopoiesis is independent of the imprinting pattern.

H19 and IGF-2 are two growth regulatory genes located on chromosome 11p15 implicated in tumorigenesis. Both genes are imprinted and regulated reciprocally under many circumstances. In order to elucidate the contribution of H19 and IGF-2 to leukemogenesis, the mRNA expression level of both genes were quantitated in bone marrow biopsies and peripheral blood samples from normal (n=98), chronic myelomonocytic leukemia (CMML, n=43), chronic myelogenous leukemia (CML, n=40) and, acute myelogenous leukemia (AML, n=32) cases. A concomitant reduction of H19 and IGF-2 expression was observed in all leukemic samples compared to the healthy controls. This down-regulation was not accompanied by changes in methylation of the differentially methylated region (DMR). Whereas the H19 gene showed strict monoallelic expression in all informative normal (n=31) and leukemic (n=54) samples, the imprinting pattern of the IGF2 gene was found to be heterogeneous. No correlations between imprinting status (mono- versus biallelic expression), quantitative mRNA expression levels and course of disease were found for the IGF-2 gene. The data suggest a disturbed regulation of the IGF-2/H19 locus in myeloid leukemias which is not caused by loss of imprinting.

Alleles↗

PubMed2004

Peptide-binding motif of HLA-A*6603.

The peptide motif of HLA-A*6603 was determined and compared with the available data on the peptide motifs of A*6601 and A*6602. A*6601 differs from A*6602 by two amino acids at positions 90 (Asp90Ala; outer loop) and 163 (Arg163Glu; pocket A). A*6603 differs from A*6601 and A*6602 by a single amino-acid exchange at position 70 (His70Gln; pockets A, B and C). No significant differences were found between the A*6602 and A*6603 peptide motifs suggesting that the Gln70His variation is of minor importance. However, the auxiliary anchors at position P1 of peptides bound by A*6601 (polar/acidic: Asp, Glu) and A*6602/6603 (polar/neutral: Ser) had striking differences. This finding may be best explained by the Arg163Glu substitution that results in a shift towards higher acidity in pocket A of A*6602/6603, apparently leading to the loss of preference for acidic auxiliary anchors. The similarity of A*6602 and A*6603 peptide motifs suggests low allogenicity when mismatched in stem cell transplantation. Inversely, the differences in A*6601 versus A*6602/6603 peptide motifs suggest that mismatches will have a higher allogenicity. These data will contribute to both assessing permissive mismatches in the A*66 group and weighting the impact of this individual amino-acid variation for matching and peptide binding algorithms.

Amino Acid Motifs↗

PubMed2004

A single amino-acid polymorphism in pocket A of HLA-A6602 alters the auxiliary anchors compared with HLA-A6601 ligands.

In this study we have sequenced peptides eluted from a truncated recombinant HLA-A*6602 molecule, and compared their features with data reported for peptides presented in the A*6601 molecule. A striking change in the amino-acid binding preferences was observed at peptide position P1, which interacts with pocket A of the HLA peptide-binding region. For A*6601, aspartic acid and glutamic acid, both of which possess polar acidic side-chains, have been described as auxiliary anchors. This is in marked contrast to A*6602, where we observed serine, which has a neutral polar side-chain, as auxiliary anchor at P1. Accordingly, this shift in the physico-chemical properties of the auxiliary anchor may be best explained by the HLA amino-acid polymorphism at position 163, where arginine (hydrophilic, alkaline) in A*6601 has been replaced by glutamic acid in A*6602. This amino-acid exchange results in a shift towards higher acidity in pocket A, apparently resulting in the loss of preference for acidic auxiliary anchors, and leading to the preference for the neutral amino acid serine. The change of the auxiliary anchor residue at P1 is likely to alter the spectrum of peptides presented by A*6602 compared with A*6601, which may result in allogenicity in the case of a mismatch in allogeneic stem cell transplantation.

Arginine↗

PubMed2003

The nature of diversity and diversification at the ABO locus.

In this study we analyzed the complete genomic sequences, except intron 1, and 2 regulatory regions of 6 common (ABO*A101, ABO*A201, ABO*B101, ABO*O01, ABO*O02, and ABO*O03) and 18 rare ABO alleles, 3 of which were new. This was done by phylogenetic analysis and correlating sequence data with the ABO phenotypes. The study revealed multiple polymorphisms in noncoding regions. The intron-based phylogenetic analysis revealed 5 main lineages: ABO*A, ABO*B, ABO*O01, ABO*O02, and ABO*O03. The genomic sequences of most rare ABO alleles differed slightly from those of the common alleles. Singular mutations or hybrid alleles were most common, but a few exhibited mosaic sequence pattern containing multiple exon and/or intron motifs from other ABO lineages. Thus, both an accumulation of mutations as well as an assortment of the mutations by recombination seems to be responsible for the ABO gene diversity. The prevalence of replacement mutations indicates positive selection for allelic diversity. Phenotype-genotype correlation showed that sequence variations within the complete coding sequence can affect A- and B-antigen expression. All variant ABO*A/B alleles and one new ABO*O03-like allele were associated with weak ABO phenotypes. These findings are suggestive of the requirement of a comprehensive coding sequence database for sequence-based phenotype prediction.

ABO Blood-Group System↗

PubMed2003

Systematic analysis of the ABO gene diversity within exons 6 and 7 by PCR screening reveals new ABO alleles.

BACKGROUND: Mutations critical for ABO blood group phenotypes have predominantly been found in exons 6 and 7 of the ABO gene, both of which encode the catalytic domain of ABO glycosyltransferase. To design rapid and reliable ABO genotyping assays, a profound knowledge of the prevalent alleles is required and a reliable sequence database needs to be established. STUDY DESIGN AND METHODS: A PCR screening system was established consisting of 102 different PCRs, each specific for a single nucleotide (nt) variation. The primer mixes were developed to walk from the 5' to the 3' end of exons 6 and 7 of the ABO gene to screen for nt mutations at 50 known polymorphic sites. A total of 109 unrelated individuals with common and rare ABO characteristics were screened. All blood samples in which the PCR results were inconclusive or inconsistent with the ABO phenotypes were subjected to sequence analysis of exons 6 and 7. RESULTS: The results of PCR screening were conclusive and consistent with the ABO phenotypes in 90 cases. In the remaining 19 cases, PCR screening revealed unusual allele combinations or amplification results that were incompatible with known ABO allele combinations or subgroups predicted by serologic analysis. In these 19 cases, sequencing revealed new ABO alleles (one ABO*Ael allele, one ABO*B(A) allele and two ABO*O alleles) in two individuals with common and seven individuals with variant ABO phenotypes. CONCLUSION: This PCR screening strategy is an effective tool for obtaining deeper insight into the ABO gene diversity and diversification and may be useful to increase the quality of the ABO sequence database.

ABO Blood-Group System↗

PubMed2003

Antibodies to high-frequency antigens may decrease the quality of transfusion support: an observational study.

BACKGROUND: There is only little information on the transfusion support of patients with antibodies to high-frequency RBC antigens. STUDY DESIGN AND METHODS: In cooperation with reference laboratories and transfusion services in Austria, Germany, and Switzerland, the transfusion support provided to hospitalized patients identified as having such antibodies was reviewed during a 20-month period. RESULTS: A total of 52 patients with antibodies to high-frequency antigens were treated in hospitals. Twenty-two of them received 104 units of antigen-negative RBCs. In 23 cases, a deviation from the standard transfusion policy (e.g., transfusion of antigen-incompatible units) occurred. The use of frozen or fresh units varied amongst the different countries but did not affect the rate of deviation from protocol. About 20 percent of all units were supplied internationally. Four antibody specificities, anti-Kpb, anti-Vel, anti-Lub, and anti-Yta, were identified in two-thirds of the patients. CONCLUSION: This survey indicated that transfusion support was unsatisfactory in about one-third of the hospitalized patients with antibodies to high-frequency antigens. Maintaining a rapidly accessible stock of just four types of rare blood units would ensure adequate transfusion support for most of these patients.

Antibody Specificity↗

PubMed2002

A weak blood group A phenotype caused by a new mutation at the ABO locus.

BACKGROUND: A number of alleles have been described for ABO encoding for common and rare ABO blood group phenotypes. Critical mutations in the coding sequence of ABO that may confer the different specificity and activity of the glycosyltransferases encoded by this gene locus have been identified. STUDY DESIGN AND METHODS: Three unrelated patients from Germany, Turkey, and Bosnia who were diagnosed as having variant A subgroups were subjected to extended ABO typing. Serologic investigations were performed with standard methods. The genetic basis of the ABO phenotypes was determined by haplotype-specific sequence analysis of the last two exons (exons 6 and 7) of ABO and the intervening intron. RESULTS: The RBCs of all three patients showed serologic A characteristics being similar to subgroup A(x). The serum of all three patients contained weakly reactive anti-A. In all three patients, sequence analysis indicated an A allele with a nucleotide sequence identical to ABO(*)A101 except for a single-base substitution in exon 7 at position 502, where C was replaced by G. This point mutation resulted in an amino acid exchange from arginine to glycine at position 168. The nucleotide sequence of intron 6 of the A allele was found to be identical to the ABO(*)A101 sequence in each patient. CONCLUSION: This study suggests that a variant A phenotype can arise from the new R168G polymorphism, reflecting the importance of this region for the ABO transferase efficiency.

ABO Blood-Group System↗